A. Claeys

The gravimetric determination of tungsten in tungstate solutions

Abstract A comparison is made between gravimetric methods for the determination of tungsten from tungstate solutions Precipitation of Hg2WO4 with Hg2(NO3)2 in ammoniacal medium is used as reference method Precipitation of WO3 with acids, followed by digestion, gives no quantitative results, the best method, using HClO4, still gives rise to a negative error larger than 1% A survey of the organic reagents is given, of these, tannin is unsatisfactory (1.2%) Better results are obtained using benzidine (0.86%), o-tolidine (0.78%), phenazone (0.77%) and rhodamine B (0.70%) Only cinchonine (0.09%) and β-naphthoquinoline (0.06%) are to be recommended for the quantitative determination of tungsten from tungstate solutions

Immobilization of glutamate decarboxylase and the preparation of an enzyme column for the synthesis of γ-[13N] aminobutyric acid

Abstract The enzyme glutamate decarboxylase ( l - glutamate 1-carboxy-lyase, EC 4.1.1.15 ) is used for the preparation of 1 μmol γ -[ 13 N ] aminobutyric acid, a radiopharmaceutical designed for positron emission tomography. To obtain a rapid and high-yield synthesis of the labelled compound, glutamate decarboxylase was immobilized on controlled pore silica beads ( pore size 50 nm, particle size 100–200 μm ), which were packed into a column. A screening of different immobilization techniques is described. The selected immobilization procedure was further optimized. The synthesis of γ -[ 13 N ] aminobutyric acid as function of enzyme loading, column dimensions and flow rate was investigated .

Direct assay for phosphotransacetylase and acetyl-coenzyme a carboxylase by high-performance liquid chromatography

A simple and specific assay to measure the activity of two coenzyme A derivative-processing enzymes, i.e., phosphotransacetylase (EC 2.3.1.8) and acetyl-coenzyme A carboxylase (EC 6.4.1.2), is described. The assay is based on the HPLC analysis of the short-chain coenzyme A derivatives formed by the enzymatic reaction, viz., acetyl-CoA and malonyl-CoA. For this purpose, ion-pair reversed-phase HPLC conditions are optimized. Furthermore, the influence of several variables on the enzyme reaction is studied in order to get maximum activity. Due to its short analysis time, good selectivity, and chromatogram information, HPLC proves to be an excellent method for the assay of these enzymes.

Assay for acetyl-CoA: arylamine N-acetyltransferase by high-performance liquid chromatography applied to serotonin N-acetylation

A specific assay to measure the activity of the enzyme acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver is described. The assay is based on the HPLC analysis of N-acetylserotonin formed by the enzymatic reaction. A reversed-phase column (Spherisorb 5-microns ODS 2; 150 x 3.2 mm) eluted with 0.1 M sodium acetate (pH 4.75)/methanol (75:25) permits baseline separation of serotonin and N-acetylserotonin within 5.3 min. Several variables on the enzyme reaction were studied to obtain maximum activity. The enzyme is most active in glycine buffer at pH 9.5. The apparent Km value for serotonin (at 0.6 mM CoASAc) is 0.246 mM and 9.9 microM for CoASAc (at 1.5 mM serotonin). To avoid acetyl-CoA or N-acetylserotonin consumption in side-reactions, the enzyme was purified. A two-step purification process (ammonium sulfate fractionation and affinity chromatography on immobilised amethopterin) yielded 60-70% of the initial enzyme activity with a purification factor of 455-560.

A new method for the pH calculation of solutions and mixtures of acids, bases and salts

Abstract The improved successive approximation method calculates the exact [H + ] and pH value of solutions and mixtures of acids, bases and salts, and can be applied to titration data. The procedure consists in the calculation of the concentration of all species present in solution, selection of the appropriate rule of electroneutrality and introduction of suitable correction factors in order to obtain a final quadratic equation which is easily solved. Compared with existing methods of pH calculation, which are based on the Newton-Raphson iteration procedure, the proposed method requires a smaller number of iterations and a shorter calculation time. As there are no limitations on the number of compounds, nor on the number or values of acidity and basicity constants, nor on the concentration range, complex problems can be solved and titration curves plotted in a short time.

Immobilization of acetylcoenzyme A synthetase and the preparation of an enzyme reactor for the synthesis of [11C]acetylcoenzyme A

The enzyme acetylcoenzyme A synthetase (acetate-CoA ligase (AMP forming), EC 6.2.1.1) from Saccharomyces cerevisiae (bakers yeast) is used for the synthesis of 1 mumol [11C]acetylcoenzyme A. (CoA-[11C]Ac). A screening of the immobilization of the enzyme on differently derivatized controlled pore glass beads (50 nm pore size and 125-180 micron particle size) was performed. Several silanes, spacer arms and terminal reactive groups were tested. The immobilized enzyme was subjected to storage stability tests. From these experiments, the method of choice was selected: immobilization on CNBr-activated controlled pore glass. The immobilized parameters were optimized further to improve the activity of the enzyme-loaded glass beads. The latter were packed in a glass column. The kinetic properties of the column were investigated and optimized to obtain an almost complete conversion of 1 mumol acetate into acetylcoenzyme A (CoA-Ac) within a few minutes. This is realized with an enzyme reactor (13.0 x 0.5 cm) containing 6.12 U active acetylcoenzyme A synthetase immobilized onto 1 g controlled pore glass.

Immobilization of human polymorphonuclear leucocyte myeloperoxidase onto controlled pore glass derivatives

Abstract To perform enzyme-catalysed bromination reactions, the enzyme myeloperoxidase (MPO; EC 1.11.1.7) from human polymorphonuclear leucocytes was immobilized onto controlled pore glass (CPG, pore size 50 nm). Different coupling techniques were screened and enzyme-loaded glass beads subjected to storage stability tests. The best results are obtained with CPG derivatized with cyanogen bromide. Different immobilization conditions were examined. Optimized enzyme immobilization was performed as follows: 0.05 U MPO, dissolved in 250 μl 0.3 m phosphate buffer pH 7.0, containing 0.3 mg ml −1 bovine serum albumin (BSA) was added to 75 mg CNBr-derivatized glass beads. The reaction mixture was kept at 0°C for 2 h, and every 10 min a shaking period of a few seconds was applied.

Purification and immobilization of acetate kinase from Desulfovibrio vulgaris

SummaryThe enzyme acetate kinase (EC 2.7.2.1) was purified fromDesulfovibrio vulgaris by a combination of ammonium sulfate precipitation, hydroxyl-apatite and dye-affinity chromatography. An overall-purification factor of 15 was obtained resulting in a specific activity of 24 U/mg protein. The purified enzyme was immobilized on differently derivatized controlled pore glass beads.

High-Performance Liquid Chromatography of α-Substituted Acetic Acid Derivatives

Abstract Two high-performance liquid chromatographic systems for the separation of α-substituted acetic acid derivatives are presented. The first method uses a resin-based column for organic acid separations (Polypore H) with a dilute acid as mobile phase. The second system decribes the possibilities of ion-pair high-performance liquid chromatography on a reverse phase C18 column. Special attention is given to the simultaneous optimization of the counterion and buffer concentration. The applicability is demonstrated in the quality control of [1-11C]-malonic acid.

Adsorption of charged substrates and products on an enzyme reactor prepared by glutaraldehyde coupling on alkylamine derivatives of Ti(IV)-coated porous silica beads

Abstract Ti(IV) coating of porous silica beads, followed by derivatization with 1,6-diaminohexane and activation with glutaraldehyde was tested for the immobilization of glutamate decarboxylase ( l -glutamate 1-carboxylyase, EC 4.1.1.15). The enzyme column prepared with the immobilized glutamate decarboxylase was designed for the preparation of 1 μmol γ-[13N]aminobutyric acid, a new tracer for positron emission tomography. Preliminary results, indicating high immobilization yields of active enzyme with good long term stabilities, led to a more detailed investigation of the Ti(IV) coating. When a column, containing about 1 g of enzyme-loaded beads was used for the synthesis of γ-[13N]aminobutyric acid (GABA) from l -[13N]glutamate, most of the13N activity remained adsorbed onto the column. The elution patterns of l -glutamate and GABA from columns of glutamate decarboxylase, immobilized on Ti(IV) coated silica beads, were investigated by using an h.p.l.c. u.v. detector. Different treatments of the Ti(IV) coated supports were tested to improve the desorption kinetics of GABA and l -glutamate. None of these methods gave a satisfactory improvement of the elution patterns of GABA and l -glutamate. The results indicate that the Ti(IV) coated silica beads have a large adsorption capacity, even though the enzyme is covalently linked. The described immobilization method is not recommended for enzymes having charged substrates or products and in which a small amount of substrate has to be applied onto a reactor containing a large amount of Ti(IV) coated support. The method can be applied when the enzyme reactor is operated in steady state conditions with continuous supply of substrate.